Eukaryotic nitric oxide synthases (NOSs) produce nitric oxide to mediate intercellular signaling and protect against pathogens. Recently, proteins homologous to mammalian NOS oxygenase domains have been found in prokaryotes and one from Bacillus subtilis (bsNOS) has been demonstrated to produce nitric oxide [Adak, S., Aulak, K. S., and Stuehr, D. J. (2002) J. Biol. Chem. 277, 16167-16171]. We present structures of bsNOS complexed with the active cofactor tetrahydrofolate and the substrate L-arginine (L-Arg) or the intermediate N(omega)-hydroxy-L-arginine (NHA) to 1.9 or 2.2 A resolution, respectively. The bsNOS structure is similar to those of the mammalian NOS oxygenase domains (mNOS(ox)) except for the absence of an N-terminal beta-hairpin hook and zinc-binding region that interact with pterin and stabilize the mNOS(ox) dimer. Changes in patterns of residue conservation between bacterial and mammalian NOSs correlate to different binding modes for pterin side chains. Residue conservation on a surface patch surrounding an exposed heme edge indicates a likely interaction site for reductase proteins in all NOSs. The heme pockets of bsNOS and mNOS(ox) recognize L-Arg and NHA similarly, although a change from Val to Ile beside the substrate guanidinium may explain the 10-20-fold slower dissociation of product NO from the bacterial enzyme. Overall, these structures suggest that bsNOS functions naturally to produce nitrogen oxides from L-Arg and NHA in a pterin-dependent manner, but that the regulation and purpose of NO production by NOS may be quite different in B. subtilis than in mammals.

The crystal structures of nitrosyl-heme complexes of a prokaryotic nitric oxide synthase (NOS) from Bacillus subtilis (bsNOS) reveal changes in active-site hydrogen bonding in the presence of the intermediate N(omega)-hydroxy-l-arginine (NOHA) compared to the substrate l-arginine (l-Arg). Correlating with a Val-to-Ile residue substitution in the bsNOS heme pocket, the Fe(II)-NO complex with both l-Arg and NOHA is more bent than the Fe(II)-NO, l-Arg complex of mammalian eNOS [Li, H., Raman, C. S., Martasek, P., Masters, B. S. S., and Poulos, T. L. (2001) Biochemistry 40, 5399-5406]. Structures of the Fe(III)-NO complex with NOHA show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound NOHA. In the Fe(II)-NO complexes, the protonated NOHA N(omega) atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. In contrast, the l-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen. This difference in hydrogen bonding to the nitrosyl group by the two substrates indicates that interactions provided by NOHA may preferentially stabilize an electrophilic peroxo-heme intermediate in the second step of NOS catalysis.

The rate of each transition was increased relative to wild-type bsNOS, with Fe III NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe II O 2 , a mixture of Fe III NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe III NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.Nitric oxide synthases (NOSs) 1 are flavoheme enzymes that generate nitric oxide (NO) from L-arginine (Arg) (1, 2). The overall reaction consumes 1.5 NADPH and 2 O 2 and involves two steps, the first being Arg hydroxylation to form N-hydroxy-L-Arg (NOHA), and the second being NOHA oxidation to form citrulline and NO (see Scheme 1).The NOS heme is located in a catalytic "oxygenase" domain that also contains the binding sites for Arg and the essential redox cofactor (6R)-tetrahydrobiopterin (H 4 B) (3, 4). The heme is ligated to a cysteine thiolate (5-7) and catalyzes a reductive activation of molecular oxygen in conjunction with H 4 B in each of the two steps of NO synthesis (8 -11). The NOS heme also binds self-generated NO as an intrinsic feature of catalysis (12)(13)(14). Each molecule of NO generated by NOS has a high probability of binding to the heme before it is released from the enzyme. The resulting ferric heme-NO product complex (Fe III NO) has been observed to build up as a transient species during NOHA oxidation reactions catalyzed by NOS oxygenase domains (NOSoxy) when run in a stopped-flow spectrophotometer under single turnover conditions (9,13,14). These experiments also provided spectral and kinetic information regarding the formation of an initial ferrous heme-dioxy species (Fe II O 2 ), whose disappearance then coincides with k cat and formation of the transient Fe III NO product complex (see Scheme 2). Because dissociation of the Fe III NO product complex is required for NO to escape from the enzyme, it is an essential step for the biologic functions of animal NOSs. In fact, this dissociation rate is one of three key kinetic parameters that together determine the overall catalytic behavior of a given NOS (15,16). Slower rates of NO dissociation dispose the Fe III NO product complex toward its reduction during catalysis, which then places the ferrous heme-NO enzyme species into a futile cycle that does not release NO (Fig. 1). Interestingly, there is only a modest variation in Fe III NO dissociation rates among the three mammalian NOS isoforms, which range from 2 to 5 s Ϫ1 when measured in NOHA single turnover reactions at 10°C (16).Given the above, we...

Estrogen has been implicated in promoting breast cancer in a majority of women. Endocrine therapy controlling estrogen production has been the guiding principle in treating breast cancer for more than a century. A greater understanding of this disease at a molecular level has led to the development of molecules that inhibit estrogen production by inhibiting the aromatase enzyme, that is the primary source of estrogen in postmenopausal women. This review examines the evolution of aromatase inhibitor (AI) based therapies over the past three decades. The third generation aromatase inhibitors (anastrozole, letrozole and exemestane), which have been found to be extremely specific and effective in an adjuvant/neoadjuvant/extended adjuvant setting are discussed from a biochemical and clinical perspective. A comprehensive discussion of the past, present, and future of aromatase inhibitors is conducted in this review.